The objective of this proposal is to develop the aromatic amino acids that have specifically incorporated 13C in their side chains. This methodology provides isolated 1H-13C spin systems for the solution structure analysis of large macromolecular systems beyond current capabilities. The new innovation is termed Pattern Specific Aromatic Labeling since isotope incorporation is at specific, known positions in the side chains of phenylalanine, tyrosine, and tryptophan. The distinct chemical shifts of aromatic acid side chains can be implemented as initiators in saturation transfer experiments and are readily distinguished in inter- and intramolecular NOE experiments. To develop a widely usable methodology, the following aims are proposed. First, the production of pattern specifically labeled phenylalanine will be optimized and the system modified to individually make pattern specific tyrosine and tryptophan. Secondly, minor modifications to the production system will be made to make pattern specifically labeled tyrosine and tryptophan. Thirdly, the incorporation of pattern specific aromatic labels into a range of recombinant proteins will be characterized. Critical attention will be paid to side chain isotope scrambling for all three aromatic residue types. The goal of these aims is to make a widely applicable, inexpensive set of reagents to explore macromolecular complexes and membrane proteins beyond current capabilities. A bacterial system has been modified to make and secrete elevated amounts of phenylalanine. Using 2-13C glycerol as the sole carbon source, C?, C?, and C? labeled phenylalanine has been made with >98% 13C incorporation. A high degree (>90%) of pattern specific labeled phenylalanine has been incorporated into recombinant ubiquitin via media supplementation. Though already less expensive than ILV methyl precursors to produce, conditions will be tested for improved yield. A range of recombinant proteins will be characterized to establish guidelines for using the methodology, including highly overepxressing ubiquitin, high aromatic content NusB, and the large thioreredoxin fusion protein scytovirin expressed in Origami bacteria. Nuclear magnetic resonance spectroscopy and mass spectrometry analysis will be used to determine isotopic labeling specificity and efficiency. Future studies include developing other specific aromatic labeling patterns and creating deuterated versions for extremely large macromolecular systems. PUBLIC HEALTH RELEVANCE: This proposal aims at developing a new set of reagents that will allow the study of macromolecular complexes and membrane proteins beyond the current state of the art capabilities. These reagents will have an impact on many aspects of human health since they have potential uses in numerous fields, particularly for the study of large macromolecular protein complexes and membrane proteins. To demonstrate the utility of the new reagents and to establish guidelines for their implementation, the researchers will apply the newly developed, inexpensive reagents to multiple recombinant systems.